The ability of lentiviruses to continually evolve and escape immune control is the central impediment in developing an effective vaccine for HIV. The lifelong persistence of virus in vivo is a function of its ability to evade immune recognition and elimination as well as its ability to replicate, i.e. it's replicative capacity. The proposed studies will use the well-characterized equine infectious anemia virus (EIAV) model to identify virus factors important in the evolution and selection of immune escape variants during progression of lentiviral disease. Retrospective samples isolated from EIAV-infected horses at sequential stages of disease will be used to test the hypothesis that virus variants which successfully evade a broadly reactive immune response contain mutations in multiple genetic regions, which confer reproducible changes in replication phenotype. The first aim will determine if env/rev genotypes that predominate at sequential stages of EIAV disease differ in replication phenotype. Recombinant infectious clones containing dominant env/rev genotypes representative of each stage of disease will be tested for replicative capacity in growth kinetic and growth competition assays. The relative fitness of pairs of variants will be estimated, and standard statistical tests used to determine when fitnesses differ significantly. These studies will establish if replication phenotype contributes to variant selection and changes in virus load during progression of disease. Subsequent aims will ascertain whether the replication differences are a cost of immune evasion, a virus strategy to evade immune recognition, or a mix of both. Studies in the second aim will determine the impact of variation in Rev and SU on replicative capacity and immune evasion. The replication phenotype of Rev will be quantified as nuclear export activity, and used to infer an immune evasion phenotype based on sensitivity to CTL killing. The SU replication phenotype will be measured as infectivity, and immune evasion phenotype determined by sensitivity to neutralizing antibody. These values, together with the replication fitness score obtained in Specific Aim 1, will be used in statistical models to determine which env/rev phenotypes affect changes in virus load during progression of disease. Experiments in the third specific aim will identify the molecular determinants of replication phenotype in variants that differ in susceptibility to broadly neutralizing antibody. EIAV-based pseudovirus will be used to map the specific regions of EIAV SU important in infectivity and immune escape identify genetic changes in SU that contribute to recrudescence of clinical disease. This detailed, integrative analyses will identify critical virus determinants that shift the balance in favor of either the virus, or the host, during progression of lentivirus disease. The design of successful vaccines for HIV and other lentiviruses requires that we understand, anticipate, and block viral strategies of evasion from a broadly reactive immune response. The information gained from the proposed studies will identify new vaccine targets that limit virus escape from broadly reactive immune responses. PROJECT NARRATIVE The proposed studies will identify the genetic mechanisms that enable lentiviruses to modify their replication and escape elimination by the host immune system. This strategy allows the virus to persist in vivo, and acquire new mutations that may increase virus load and lead to progression of clinical disease. The results of this study will identify new vaccine strategiesw for HIV-1 that inhibit the viruses ability to escape from the host immune response.